Development of innovative statistical approaches to biological product risk and decision analysis. This project was conceived, directed and conducted by Rolf Taffs, PhD. The goal of the project is to advance the FDA's mission to protect consumers by providing scientifically sound criteria to assure the safety and potency of vaccines and immune globulins, to prevent the transmission of viruses by blood, and to assess the risks associated with transmissible spongiform encephalopathies and regulated products. The project is intended to provide CBER and other Centers in the FDA with valid methods and statistical approaches that bridge the fields of biostatistics and laboratory-based experimental microbiology and immunology. Earlier research on vaccine test development resulted in development of statistical test-validity criteria and decision (lot release) criteria that were accepted by WHO Working Parties and the WHO Expert Committee on Biological Standards for two alternate tests for the safety of Poliovirus Vaccine Live Oral (OPV): 1) Mutant Analysis by PCR and Restriction Endonuclease Cleavage (MAPREC), and 2) the Transgenic Mouse Neurovirulence test (v.i.). In addition an innovative test was developed for the potency of inactivated poliovirus vaccine based on protection of transgenic mice susceptible to polioviruses against lethal challenge with wild-type viruses. Other more recent collaborative efforts involving nine CBER laboratories in OBRR, OVRR, and OTRR (CDER) include the following: 1. development of the statistical basis for a test to predict the neurovirulence of mumps vaccines; 2. an assessment of inhibition of immune response to inactivated polioviruses by other components of combined vaccines; 3. research on the modulation of susceptibility of macrophages to infection with HIV and apoptosis in infected monocytes and cell lines; 4. development of accessible statistically sound models for validation and analysis of data generated by oligonucleotide microarrays to discriminate viral subtypes and for improved immunoassays that may offer more sensitive screening of blood donors infected with human T-cell lymphotropic viruses; 5. provided guidance for development of a new national reference standard for Rho(D) immune globulin; 6. provided guidance on preparing quantitative risk analyses for transmissible spongiform encephalopathies based on the most current statistically sound principles (based on advanced training in quantitative risk assessment techniques received from the Harvard School of Public Health and Palisades Corp.); 7. produced risk models and sensitivity analyses for Creutzfeldt-Jakob disease (CJD) in CBER-regulated transplanted tissues; 8. with CDRH scientists, development of an analytical approach suitable for addressing the risk of transmitting CJD by FDA-regulated surgical instruments and implanted devices. 9. developed statistical methods for evaluation of a novel vaccinia neutralization assay based on a reporter gene expression; 10. provided guidance on statistical approaches for the development and validation of pre-clinical toxicity assays for vaccinia-based smallpox vaccines. Test to detect residual spongiform encephalopathy agents on environmental surfaces. The infectious agents causing transmissible spongiform encephalopathies (TSE agents or prions) are notoriously difficult to inactivate completely. Previous studies indicated that drying infected tissues increased the resistance of the TSE agents to inactivation. We modified a test originally designed to evaluate effects of disinfectants on conventional viruses to detect residual infectivity remaining on glass surfaces after various treatments intended to inactivate or remove TSE agents (using the 263K strain of hamster-adapted scrapie agent as a model). The modified method employs only disposable equipment to reduce opportunities for cross contamination. Preliminary studies with suspensions of conventional viruses showed little within-test variability but significant between-test variability, so that a reference sham-treated preparation of scrapie-infected tissue suspension was included and titrated in each test. Control treatments with diluents containing no disinfectants removed only negligible amounts of scrapie infectivity from surfaces. As previously observed, residual scrapie infectivity was detected after autoclaving at 134 C for 1 hr. Recent results demonstrated that treatments with NaOH, NaOCl and concentrated formic acid were more effective. A variety of other decontamination regimens are under study, including detergents, phenolic disinfectants and combinations of agents with and without autoclaving, and ultrasonic cleaning in a variety of solutions. With investigators at CDRH, this project has been expanded to examine decontamination of needles made of various metals and plastic-coated needles exposed to the same strain of hamster-adapted scrapie agent and assayed as described. Additional funding to support the project has was provided by the FDA Office of Science. This project is to be continued in FY 04. Bacterial Contaminants of Blood and Blood Products. Transfusion-associated septic reactions (TAS), that occur due to transfusion of blood and blood components contaminated with bacteria are often fatal. Red blood cells (RBC), which are stored at 4 C, are most frequently contaminated with Gram-negative bacteria such as Yersinia enterocolitica, Serratia liquefaciens, Pseudomonas fluorescens, etc. Bacteria associated with platelet concentrates, which are stored at room temperature, are most often skin bacteria: Gram-positive cocci--such as Staphylococcus epidermidis and Staphylococcus aureus--Bacillus cereus, and Gram-negative bacteria such as Enterobacter cloacae, Klebsiella spp and Serratia marcescens. Methods to detect small numbers of these bacteria rapidly are lacking, as is knowledge of the true incidence rate of bacterial contamination of blood products. Existing methods require concentrations of about 10(4) to 10(7) bacteria /ml, before the bacteria can be detected. Nucleic-acid-based testing for detection, such as the polymerase chain reaction (PCR) is extremely sensitive and has the theoretical potential of detecting one bacterium in a sample within hours. Moreover it can identify the species of contaminating bacteria. Investigators in LBPUA developed a highly sensitive PCR assay for detecting Y. enterocolitica in blood samples stored up to 42 days at 4 C. (Y. enterocolitica accounts for 50% of the clinical sepsis caused by RBC contamination.) The method used is a fluorogenic 5'-nuclease assay, (the TaqMan PCR assay), which has the following advantages: (1) It is a "real-time" PCR assay using a fluorogenic oligonucleotide probe in the reaction. Thus, it has the potential of detecting a target nucleic acid sequence in less than 2 hours. (2) It is a highly quantitative PCR that can indicate numbers of copies of a gene present in a unknown sample. (3) It easily lends itself to automation. LBPUA investigators designed primers and a probe targeted to the 16S rRNA gene region of Y. enterocolitica. There are several copies of this gene per cell, and during active cell growth the abundance of the rRNA can be as high as 10(4) copies per cell, meaning that fewer target bacteria are needed to detect a contaminated unit. Several methods to purify chromosomal DNA from blood spiked with bacteria were evaluated; hemoglobin, which is a potent inhibitor of Taq polymerase must be efficiently removed, and extraction reagents must not interfere with the fluorescence. The sensitivity achieved with the TaqMan PCR assay for Y. enterocolitica was 30 bacteria per ml of blood. The primers and probe are specific for Y. enterocolitica and did not detect any other Yersinia species. In a second phase of the project the Y. enterocolitica assay model was used to detect a broader spectrum of contaminating bacteria: Yersinia, Serratia, Klebsiella and Enterobacter. The rRNA gene has conserved DNA regions, interspersed with variable sequences, in related bacteria. Thus a "universal" forward and reverse primer set directed towards the conserved regions shared by these bacteria species was designed. Two probes, one specific for all Yersinia species and a second probe specific for all Serratia, Klebsiella and Enterobacter species reported to contaminate blood were used in a single multiplex TaqMan PCR assay. The DNA extraction method was modified slightly, and the sensitivity achieved was 12-16 bacteria per ml of whole blood. In the next phase of the project, now in progress, primers and probes have been selected from the 16S rRNA region of 17 bacteria that most commonly contaminate platelets. Platelets are seeded with these bacteria and the total DNA extracted from these samples and tested in a single multiplex TaqMan assay. Progress in this project has resumed under the direction of Sanjai Kumar, PhD. Another investigator has been recruited to join LBPUA and to continue this research during the coming fiscal year. Susceptibility of genetically modified candidate cell substrates to infections with TSE agents. Assessment of the risk of mutations in the PrP gene associated with familial Creutzfeldt-Jakob disease: implications for the safety of cell substrates used to prepare vaccines and other biologics Background: Agents of transmissible spongiform encephalopathies (TSE) must be considered as potential adventitious agents in biologics. There have been precedents when vaccines accidentally prepared from tissues of scrapie-infected sheep caused scrapie outbreaks involving hundreds of immunized animals. Emergence of new-variant Creutzfeldt-Jakob disease (vCJD) and its probable link to bovine spongiform encephalopathy (BSE) made this an important issue for human vaccines and other biologics as well. The risk is best addressed by careful selection of source materials used to produce biologics. However, there is another hypothetical mechanism by which human biologics produced in cell cultures can become contaminated with TSE agents. According to the most widely held opinion (the "prion" hypothesis, commonly though not universally accepted), TSE agents can emerge de novo by aberrant folding of a normal protein produced by a cellular gene--the prion protein or PrP. Such transition from a normal protein to its infectious form (associated with an increase in beta-pleated-sheet content, loss of solubility in detergent solutions and markedly increased resistance to digestion by the enzyme proteinase K) is thought to be facilitated by any of a number of point mutations or reduplications in the PrP-encoding gene. These mutations are consistently found in one of the two PrP genes of patients with various familial forms of CJD, which is inherited as an autosomal dominant trait of variable penetrance depending on the specific mutation involved. Since cell cultures used as substrates to prepare biologics are serially propagated in vitro in extremely large numbers, there is a theoretical but troubling possibility that spontaneous mutation in the PrP gene might result in contamination of vaccines with infectious protein. Although this scenario is purely hypothetical, it cannot be discounted until experimental evaluation of infectivity of the mutant PrP gene products is performed. Rationale and experimental design: To prove that spontaneous PrP gene mutations that may occur in candidate cell substrates pose no risk for vaccine recipients, we engineered a line of human cells (SY5Y) capable of expressing a variety of neuronal proteins to express recombinant PrP genes bearing one or another of the three point mutations most often found in patients with familial CJD (fCJD)and propagated. Evaluation of the cultures by fluorescence-activated cell sorting showed high expression of prion proteins on the cell surface in comparison with untransfected cells of the same line. Cells will then be sequenced to confirm the continued presence of the fCJD-associated mutations and tested for the presence of the protease-resistant PrP that typically appears in tissues of humans and animals with TSEs. To determine whether actual infectivity has appeared during serial passaging of the cells bearing the fCJD-associated mutations, extracts from large numbers of these cells will be inoculated intracerebrally into squirrel monkeys--the most sensitive validated model for detection of human TSE agents; monkeys will be observed for at least five years for evidence of TSE. (If an adequate number of transgenic mice become available, cells may be assayed for infectivity in mice as well.) Funding for this part of the project has been granted by NIAID and animals obtained. Furthermore, even if the mutant proteins are not themselves infectious, their presence might increase susceptibility of cell substrates to infections with a TSE agent present in the environment. Therefore, should additional funds be obtained, the same recombinant cultures may be exposed to small amounts of reference CJD agent, propagated for a number of passages sufficient to eliminate all of the remaining original inoculum, and then assayed for genetic stability, protease-resistant PrP and infectivity as described above. Two results are possible. If no monkey develops disease, then we may conclude that, under conditions that highly favored replication of the TSE agent, no infectivity appeared, implying that the risk for recipients of biologics prepared in the unmutated cell substrate must be exceedingly small. But if any monkey develops a TSE, then the substrate must be considered to pose a potential threat. (The result might also provide at least circumstantial support for or against the "prion" hypothesis of spontaneous self-propagating changes in protein folding as the basis of TSE infectivity.) In a related study, we examined the intrinsic stability of the prion-protein-encoding gene (PRNP gene)in HeLa cells--the human cell line with the longest known history of propagation in culture. HeLa cells contain a well-known deletion in the PRNP gene--a deletion also found in about 2% of normal people. The analysis of that deletion in various lineages of HeLa cells suggests that the progenitor of several popular lines lost all or part of a chromosome early in the history of HeLa cells. No other deletions, duplications or point mutations were found in these extensively passaged cell lines, showing that the PRNP gene has been very stable over many hundreds of passages in culture and that the spontaneous emergence of mutated prion protein molecules in continuously passaged heteroploid cell substrates used for vaccine production is not likely. (This work was recently published.) This project incorporates FY2002 projects 1Z01BP005001-04, 1Z01BP005002-03, 1Z01BP005006-06, 1Z01BP005017-02, and 1Z01BP005020-02.